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A long-standing challenge is to understand at the atomic level how protein dynamics contribute to enzyme catalysis. X-ray crystallography can provide snapshots of conformational substates sampled during enzymatic reactions, while NMR relaxation methods reveal the rates of interconversion between substates and the corresponding relative populations. However, these current methods cannot simultaneously reveal the detailed atomic structures of the rare states and rationalize the finding that intrinsic motions in the free enzyme occur on a timescale similar to the catalytic turnover rate. Here we introduce dual strategies of ambient-temperature X-ray crystallographic data collection and automated electron-density sampling to structurally unravel interconverting substates of the human proline isomerase, cyclophilin A (CYPA, also known as PPIA). A conservative mutation outside the active site was designed to stabilize features of the previously hidden minor conformation. This mutation not only inverts the equilibrium between the substates, but also causes large, parallel reductions in the conformational interconversion rates and the catalytic rate. These studies introduce crystallographic approaches to define functional minor protein conformations and, in combination with NMR analysis of the enzyme dynamics in solution, show how collective motions directly contribute to the catalytic power of an enzyme. [ABSTRACT FROM AUTHOR]

Examines the packaging of cyclophilin A (CypA) for efficient replication. Visualization of catalytic activity by nuclear magnetic resonance exchange spectroscopy; Observation on the CypA binding and catalysis; Conformation on the heterogeneity for prolyl peptide bond in the folded state.

Reversible protein phosphorylation directs essential cellular processes including cell division, cell growth, cell death, inflammation, and differentiation. Because protein phosphorylation drives diverse diseases, kinases and phosphatases have been targets for drug discovery, with some achieving remarkable clinical success. Most protein kinases are activated by phosphorylation of their activation loops, which shifts the conformational equilibrium of the kinase toward the active state. To turn off the kinase, protein phosphatases dephosphorylate these sites, but how the conformation of the dynamic activation loop contributes to dephosphorylation was not known. To answer this, we modulated the activation loop conformational equilibrium of human p38a MAP kinase with existing kinase inhibitors that bind and stabilize specific inactive activation loop conformations. From this, we identified three inhibitors that increase the rate of dephosphorylation of the activation loop phospho-threonine by the PPM serine/threonine phosphatase WIP1. Hence, these compounds are "dual-action" inhibitors that simultaneously block the active site and promote p38a dephosphorylation. Our X-ray crystal structures of phosphorylated p38a bound to the dual-action inhibitors reveal a shared flipped conformation of the activation loop with a fully accessible phospho-threonine. In contrast, our X-ray crystal structure of phosphorylated apo human p38a reveals a different activation loop conformation with an inaccessible phospho-threonine, thereby explaining the increased rate of dephosphorylation upon inhibitor binding. These findings reveal a conformational preference of phosphatases for their targets and suggest a unique approach to achieving improved potency and specificity for therapeutic kinase inhibitors. [ABSTRACT FROM AUTHOR]

Protein language models (pLMs) have emerged as potent tools for predicting and designing protein structure and function, and the degree to which these models fundamentally understand the inherent biophysics of protein structure stands as an open question. Motivated by a finding that pLM-based structure predictors erroneously predict nonphysical structures for protein isoforms, we investigated the nature of sequence context needed for contact predictions in the pLM Evolutionary Scale Modeling (ESM-2). We demonstrate by use of a "categorical Jacobian" calculation that ESM-2 stores statistics of coevolving residues, analogously to simpler modeling approaches like Markov Random Fields and Multivariate Gaussian models. We further investigated how ESM-2 "stores" information needed to predict contacts by comparing sequence masking strategies, and found that providing local windows of sequence information allowed ESM-2 to best recover predicted contacts. This suggests that pLMs predict contacts by storing motifs of pairwise contacts. Our investigation highlights the limitations of current pLMs and underscores the importance of understanding the underlying mechanisms of these models. [ABSTRACT FROM AUTHOR]

Despite the outstanding success of the cancer drug imatinib, one obstacle in prolonged treatment is the emergence of resistance mutations within the kinase domain of its target, Abl. We noticed that many patient-resistance mutations occur in the dynamic hot spots recently identified to be responsible for imatinib’s high selectivity toward Abl. In this study, we provide an experimental analysis of the mechanism underlying drug resistance for three major resistance mutations (G250E, Y253F, and F317L). Our data settle controversies, revealing unexpected resistance mechanisms. The mutations alter the energy landscape of Abl in complex ways: increased kinase activity, altered affinity, and cooperativity for the substrates, and, surprisingly, only a modestly decreased imatinib affinity. Only under cellular adenosine triphosphate (ATP) concentrations, these changes cumulate in an order of magnitude increase in imatinib’s half-maximal inhibitory concentration (IC50). These results highlight the importance of characterizing energy landscapes of targets and its changes by drug binding and by resistance mutations developed by patients. [ABSTRACT FROM AUTHOR]

To study microsecond processes by relaxation dispersion NMR spectroscopy, low power deposition and short pulses are crucial and encourage the development of experiments that employ ±H Carr-Purcell-Meiboom-Gill (CPMG) pulse trains. Herein, a method is described for the comprehensive study of microsecond to millisecond time scale dynamics of methyl groups in proteins, exploiting their high abundance and favorable relaxation properties. In our approach, protein samples are produced using [±H, 13C]-d-glucose in 100% D2O, which yields CHD2 methyl groups for alanine, valine, threonine, isoleucine, leucine, and methionine residues with high abundance, in an otherwise largely deuterated background. Methyl groups in such samples can be sequence-specifically assigned to near completion, using 13C TOCSY NMR spectroscopy, as was recently demonstrated (Otten, R.; et al. J. Am. Chem. Soc. 2010, 132, 2952-2960). In this Article, NMR pulse schemes are presented to measure ¹H CPMG relaxation dispersion profiles for CHD2 methyl groups, in a vein similar to that of backbone relaxation experiments. Because of the high deuteration level of methyl-bearing side chains, artifacts arising from proton scalar coupling during the CPMG pulse train are negligible, with the exception of Ile-δ1 and Thr-γ2 methyl groups, and a pulse scheme is described to remove the artifacts for those residues. Strong 13C scalar coupling effects, observed for several leucine residues, are removed by alternative biochemical and NMR approaches. The methodology is applied to the transcriptional activator NtrCr, for which an inactive/active state transition was previously measured and the motions in the microsecond time range were estimated through a combination of backbone 15N CPMG dispersion NMR spectroscopy and a collection of experiments to determine the exchange-free component to the transverse relaxation rate. Exchange contributions to the ¹H line width were detected for 21 methyl groups, and these probes were found to collectively report on a local structural rearrangement around the phosphorylation site, with a rate constant of (15.5 ± 0.5) × 10³ per second (i.e., τex = 64.7 ± 1.9 μs). The affected methyl groups indicate that, already before phosphorylation, a substantial, transient rearrangement takes place between helices 3 and 4 and strands 4 and 5. This conformational equilibrium allows the protein to gain access to the active, signaling state in the absence of covalent modification through a shift in a pre-existing dynamic equilibrium. Moreover, the conformational switching maps exactly to the regions that differ between the solution NMR structures of the fully inactive and active states. These results demonstrate that a cost-effective and quantitative study of protein methyl group dynamics by ¹H CPMG relaxation dispersion NMR spectroscopy is possible and can be applied to study functional motions on the microsecond time scale that cannot be accessed by backbone 15N relaxation dispersion NMR. The use of methyl groups as dynamics probes extends such applications also to larger proteins. [ABSTRACT FROM AUTHOR]

Despite being the subject of intense effort and scrutiny, kinases have proven to be consistently challenging targets in inhibitor drug design. A key obstacle has been promiscuity and consequent adverse effects of drugs targeting the ATP binding site. Here we introduce an approach to controlling kinase activity by using monobodies that bind to the highly specific regulatory allosteric pocket of the oncoprotein Aurora A (AurA) kinase, thereby offering the potential for more specific kinase modulators. Strikingly, we identify a series of highly specific monobodies acting either as strong kinase inhibitors or activators via differential recognition of structural motifs in the allosteric pocket. X-ray crystal structures comparing AurA bound to activating vs inhibiting monobodies reveal the atomistic mechanism underlying allosteric modulation. The results reveal 3 major advantages of targeting allosteric vs orthosteric sites: extreme selectivity, ability to inhibit as well as activate, and avoidance of competing with ATP that is present at high concentrations in the cells. We envision that exploiting allosteric networks for inhibition or activation will provide a general, powerful pathway toward rational drug design. [ABSTRACT FROM AUTHOR]

Summary: Synaptic plasticity induced by cocaine and other drugs underlies addiction. Here we elucidate molecular events at synapses that cause this plasticity and the resulting behavioral response to cocaine in mice. In response to D1-dopamine-receptor signaling that is induced by drug administration, the glutamate-receptor protein metabotropic glutamate receptor 5 (mGluR5) is phosphorylated by microtubule-associated protein kinase (MAPK), which we show potentiates Pin1-mediated prolyl-isomerization of mGluR5 in instances where the product of an activity-dependent gene, Homer1a, is present to enable Pin1-mGluR5 interaction. These biochemical events potentiate N-methyl-D-aspartate receptor (NMDAR)-mediated currents that underlie synaptic plasticity and cocaine-evoked motor sensitization as tested in mice with relevant mutations. The findings elucidate how a coincidence of signals from the nucleus and the synapse can render mGluR5 accessible to activation with consequences for drug-induced dopamine responses and point to depotentiation at corticostriatal synapses as a possible therapeutic target for treating addiction. [ABSTRACT FROM AUTHOR]

Internal protein dynamics are intimately connected to enzymatic catalysis. However, enzyme motions linked to substrate turnover remain largely unknown. We have studied dynamics of an enzyme during catalysis at atomic resolution using nuclear magnetic resonance relaxation methods. During catalytic action of the enzyme cyclophilin A, we detect conformational fluctuations of the active site that occur on a time scale of hundreds of microseconds. The rates of conformational dynamics of the enzyme strongly correlate with the microscopic rates of substrate turnover. The present results, together with available structural data, allow a prediction of the reaction trajectory. [ABSTRACT FROM AUTHOR]

Studies the two-state allosteric behavior in a single-domain signaling protein. Correlationship between phosphorylation-driven activation of the signaling protein NtrC and microsecond time-scale backbone dynamics; Stabilization of preexisting conformations; Description of a significant fraction of active molecules before phosphorylation.

How can a single protein domain encode a conformational landscape with multiple stably folded states, and how do those states interconvert? Here, we use real-time and relaxation-dispersion NMR to characterize the conformational landscape of the circadian rhythm protein KaiB from Rhodobacter sphaeroides . Unique among known natural metamorphic proteins, this KaiB variant spontaneously interconverts between two monomeric states: the "Ground" and "Fold-switched" (FS) states. KaiB in its FS state interacts with multiple binding partners, including the central KaiC protein, to regulate circadian rhythms. We find that KaiB itself takes hours to interconvert between the Ground and FS state, underscoring the ability of a single-sequence to encode the slow process needed for function. We reveal the rate-limiting step between the Ground and FS state is the cis-trans isomerization of three prolines in the fold-switching region by demonstrating interconversion acceleration by the prolyl isomerase Cyclophilin A. The interconversion proceeds through a "partially disordered" (PD) state, where the C-terminal half becomes disordered while the N-terminal half remains stably folded. We found two additional properties of KaiB's landscape. First, the Ground state experiences cold denaturation: At 4 °C, the PD state becomes the majorly populated state. Second, the Ground state exchanges with a fourth state, the "Enigma" state, on the millisecond-timescale. We combine AlphaFold2-based predictions and NMR chemical shift predictions to predict this Enigma state is a beta-strand register shift that relieves buried charged residues, and support this structure experimentally. These results provide mechanistic insight into how evolution can design a single-sequence that achieves specific timing needed for its function., Competing Interests: Competing interests statement:D.K. is a co-founder of Relay Therapeutics and MOMA Therapeutics. The remaining authors declare no competing interests.

Reversible protein phosphorylation directs essential cellular processes including cell division, cell growth, cell death, inflammation, and differentiation. Because protein phosphorylation drives diverse diseases, kinases and phosphatases have been targets for drug discovery, with some achieving remarkable clinical success. Most protein kinases are activated by phosphorylation of their activation loops, which shifts the conformational equilibrium of the kinase towards the active state. To turn off the kinase, protein phosphatases dephosphorylate these sites, but how the conformation of the dynamic activation loop contributes to dephosphorylation was not known. To answer this, we modulated the activation loop conformational equilibrium of human p38α ΜΑP kinase with existing kinase inhibitors that bind and stabilize specific inactive activation loop conformations. From this, we discovered three inhibitors that increase the rate of dephosphorylation of the activation loop phospho-threonine by the PPM serine/threonine phosphatase WIP1. Hence, these compounds are "dual-action" inhibitors that simultaneously block the active site and stimulate p38α dephosphorylation. Our X-ray crystal structures of phosphorylated p38α bound to the dual-action inhibitors reveal a shared flipped conformation of the activation loop with a fully accessible phospho-threonine. In contrast, our X-ray crystal structure of phosphorylated apo human p38α reveals a different activation loop conformation with an inaccessible phospho-threonine, thereby explaining the increased rate of dephosphorylation upon inhibitor binding. These findings reveal a conformational preference of phosphatases for their targets and suggest a new approach to achieving improved potency and specificity for therapeutic kinase inhibitors., Competing Interests: Competing interests N.B. and E.S. are the inventors on a pending patent on a new method for optimizing kinase inhibitors applied for by Brandeis University. D.K. is co-founder of Relay Therapeutics and MOMA Therapeutics. The remaining authors declare no competing interests.